Open AccessResearch Role of HIV-1 subtype C envelope V3 to V5 regions in viral entry, coreceptor utilization and replication efficiency in primary T-lymphocytes and monocyte-derived ma
Trang 1Open Access
Research
Role of HIV-1 subtype C envelope V3 to V5 regions in viral entry,
coreceptor utilization and replication efficiency in primary
T-lymphocytes and monocyte-derived macrophages
Vasudha Sundaravaradan†1, Suman R Das†2,4, Rajesh Ramakrishnan1,5,
Shobha Sehgal3, Sarla Gopalan3, Nafees Ahmad*1 and Shahid Jameel*2
Address: 1 Department of Immunobiology, College of Medicine, University of Arizona, Tucson, AZ 85724, USA, 2 Virology Group, International Center for Genetic Engineering and Biotechnology, New Delhi, India, 3 Departments of Pathology and Obstetrics and Gynecology, Post Graduate Institute of Medical Education and Research, Chandigarh, India, 4 NIAID, National Institutes of Health, Bethesda, MD 20892, USA and
5 Department of Molecular Virology & Microbiology, Baylor College of Medicine, Houston, TX 77030, USA
Email: Vasudha Sundaravaradan - vsvaradan@yahoo.com; Suman R Das - dassr@niaid.nih.gov; Rajesh Ramakrishnan - ramakris@bcm.tmc.edu; Shobha Sehgal - sehgal@hotmail.com; Sarla Gopalan - Gopalan@hotmail.com; Nafees Ahmad* - nafees@u.arizona.edu;
Shahid Jameel* - jameelshahid@gmail.com
* Corresponding authors †Equal contributors
Abstract
Background: Several subtypes of HIV-1 circulate in infected people worldwide, including subtype
B in the United States and subtype C in Africa and India To understand the biological properties
of HIV-1 subtype C, including cellular tropism, virus entry, replication efficiency and cytopathic
effects, we reciprocally inserted our previously characterized envelope V3–V5 regions derived
from 9 subtype C infected patients from India into a subtype B molecular clone, pNL4-3 Equal
amounts of the chimeric viruses were used to infect T-lymphocyte cell lines (A3.01 and MT-2),
coreceptor cell lines (U373-MAGI-CCR5/CXCR4), primary blood T-lymphocytes (PBL) and
monocyte-derived macrophages (MDM)
Results: We found that subtype C envelope V3–V5 region chimeras failed to replicate in
T-lymphocyte cell lines but replicated in PBL and MDM In addition, these chimeras were able to infect
U373MAGI-CD4+-CCR5+ but not U373MAGI-CD4+-CXCR4+ cell line, suggesting CCR5
coreceptor utilization and R5 phenotypes These subtype C chimeras were unable to induce
syncytia in MT-2 cells, indicative of non-syncytium inducing (NSI) phenotypes More importantly,
the subtype C envelope chimeras replicated at higher levels in PBL and MDM compared with
subtype B chimeras and isolates Furthermore, the higher levels subtype C chimeras replication in
PBL and MDM correlated with increased virus entry in U373MAGI-CD4+-CCR5+
Conclusion: Taken together, these results suggest that the envelope V3 to V5 regions of subtype
C contributed to higher levels of HIV-1 replication compared with subtype B chimeras, which may
contribute to higher viral loads and faster disease progression in subtype C infected individuals than
other subtypes as well as rapid HIV-1 subtype C spread in India
Published: 24 November 2007
Virology Journal 2007, 4:126 doi:10.1186/1743-422X-4-126
Received: 9 October 2007 Accepted: 24 November 2007
This article is available from: http://www.virologyj.com/content/4/1/126
© 2007 Sundaravaradan et al; licensee BioMed Central Ltd
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Trang 2The steepest increase in new cases of human
immunode-ficiency virus type 1 (HIV-1) infection has taken place in
South America [1] and South/Southeast Asia [2], of which
India is experiencing a rapid and extensive spread of
infec-tion National surveys in India have shown that the spread
in India is primarily heterosexual in the metropolitan and
coastal cities, and via intravenous drug use in the
North-east bordering Myanmar [3,4] The HIV-1 sequences
ana-lyzed from different cohorts from several regions in India
suggests that HIV-1 subtype C is the predominant subtype
found in India [3-8] It has also been shown in several
African and South American studies that subtype C
rap-idly predominates over all the other HIV-1 subtypes after
being introduced in those populations [1,9], suggesting
that subtype C may soon become the prevalent subtype
worldwide However, the biological properties of HIV-1
subtype C viruses that may influence its rapid spread are
not known
HIV-1 envelope gp120 interacts with CD4 receptor and
CXCR4 or CCR5 coreceptor [10-13] on T lymphocytes,
monocytes/macrophages and other cell types [11,14,15]
to enter target cells Analysis of env gp120 sequences from
a large number of HIV-1 isolates shows that gp120 is
made up of five variable regions (V1 to V5) that are
inter-spersed with conserved regions [16] The potential
patho-genic region of HIV-1 presumably lies within these
variable regions, especially in the V3 region comprising of
35 amino acids arranged in a disulphide loop involving
two cysteines [10] The hypervariable region 3, the V3
region, is functionally important in virus infectivity
[17-19], virus neutralization [13,20-22], replication efficiency
and host cell tropism [10,23], whereas the V1–V2 regions
influence replication efficiency in macrophages by
affect-ing virus spread [24,25] In addition, the variable loops V4
and V5 of gp120 are less flexible regions of the proteins
and may play roles in CD4 binding and neutralizing
anti-body responses [26,27] The mechanisms by which the V3
domain and other regions of the env glycoprotein control
cell tropism were described by identifying two distinct
co-receptors, fusin (CXR4) and CCR5, for the entry of
T-lym-photropic and macrophage-tropic HIV-1, respectively
[11,15] The region responsible for determining
corecep-tor utilization was examined by Choe et al., [12] and
showed that the V3 region was responsible for interacting
with this co-receptor Several studies have shown that a
reciprocal transfer of an HIV-1 R5 clones' V3 region into
an X4 molecular clone changed its tropism to allow
infec-tion and replicainfec-tion in macrophages [10,19,23,28-30]
However, most of the data on viral infectivity, coreceptor
utilization, replication efficiency and cytopathic effects
have been obtained from HIV-1 subtype B, and very
lim-ited information is available on subtype C viruses,
espe-cially those from India
In this study, we have characterized the biological proper-ties of HIV-1 subtype C envelope V3 to V5 regions by con-structing chimeric recombinant viruses containing subtype C envelope V3–V5 regions from nine infected patients from India [5] into subtype B infectious molecu-lar clone, pNL4-3 We show that the envelope V3–V5 regions of HIV-1 subtype C changed the tropism of HIV-1 NL4-3 from X4 to R5 and contributed to the increased virus entry and replication efficiency in primary blood T-lymphocytes (PBL) and monocyte-derived macrophages (MDM) compared with subtype B viruses This higher rep-lication efficiency of subtype C compared with subtype B may contribute to a higher viral load and faster disease progression in patients infected with HIV-1 subtype C viruses in the Indian population
Results
Characterization and comparison of subtype C envelope V3–V5 chimeras' sequences with known isolates
We confirmed the reciprocal insertion of the sixteen enve-lope V3–V5 region sequences (Fig 1) from nine subtype
C infected patients' isolates from India (Table 1) that were sequenced before [5] into pNL4-3 by nucleotide sequenc-ing Two clones were selected from each patient for recip-rocal insertion The patients harbored various stages of HIV disease (I to IVE) based on the 1987 CDC
classifica-tion We also compared the subtype C env V3 to V5
regions chimeras with HIV-1NL4-3, HIV-1BaL and a known
env V3–V5 region sequence of subtype C (Fig 1) All the
subtype C env V3–V5 clones showed a greater similarity to
the R5 virus HIV-1BaL than HIV-1NL4-3 The amino acids critical for the R5 phenotype, including the Y at position
283 and E or D at position 287 were present in all subtype
C clones The amino acid H at position 275 seen in
HIV-1Ba-L which is also important for determining R5 tropism was present only in the clones from patient 17 (clones 171 and 173) The amino acid sequences between positions 310–315 and 350–373 were significantly different from HIV-1NL4-3 clone but were very similar to the previously known sequence of subtype C envelope region It is inter-esting to note that a critical glycosylation site, which includes the first cysteine of the V3 loop, was mutated in all the clones except those obtained from patient 17 and
5 (clones 171, 173, 512, 514) The sequence analysis of all the clones indicate that these clones are different from HIV-1NL4-3 envelope sequences but very similar to enve-lope sequences from known subtype C clones and to R5 tropic viruses such as HIV-1BaL
Computational prediction of coreceptor usage using V3 amino acid sequence of subtype C chimeras
We also used the Position Specific Scoring Matrix (PSSM) bioinformatics tool (X4/R5) to predict coreceptor usage
by the Subtype C patient env V3–V5 region sequences [31]
and calculated scores for each clone are shown in Table 2
Trang 3Table 1: Patient demographics, possible source and risk factor of transmission and CDC disease classification.
Patient Code Chimeras Number Age/Sex Possible source Risk factor CDC classification
heterosexual
IV-C, D
heterosexual
II
heterosexual
IV-C
heterosexual
II
CDC-Center for Disease Control and Prevention
Comparison of envelope (V3 to V5 regions) from subtype C chimeras with subtype B (X4 and R5) and C envelope sequences
Figure 1
Comparison of envelope (V3 to V5 regions) from subtype C chimeras with subtype B (X4 and R5) and C envelope sequences
The sequences of subtype C env chimeras used in this study were analyzed by performing multiple sequence alignment with
parental clone HIV-1NL4-3 as a reference and HIV-1BaL and known HIV-1 subtype C envelope regions for comparison Subtype C chimeras are designated by numbers Dots indicate a match with the reference sequence whereas substitutions are indicated
by the single letter code for the changed amino acid Gaps are shown as dashes Structural elements of the envelope are indi-cated by spanning arrowheads and glycosylation sites are indiindi-cated by asterisk Amino acid positions are indiindi-cated to denote the amino acid numbers of the complete envelope gp120
*** *** V3 *** *** ***
NL43 RSANFTDNAK TIIVQLNTSV EINCTRPNNN TRKSIRIQRG PGRAFVTIGK -IGNMRQAHC NISRAKWNAT LKQIASKLRE QFGNNKTIIF KQSSGGDPEI VTHSFNC BAL E N E .H. .Y.T.E I DI L D .NK.VI .- V .H
171 E.L.N VEP T H. Q YAT.D L DT.K.Y TVNGTN R .HKV.EQ.GR H. .R N TKP L T
173 E.L.N VEP T H. Q YAT.D L DT.K.Y TVNGTN R .HKV.EQ.GR H. .R N TKP L T
182 E.L V H.DQP V.I V R QT.YAT.D I DI.K.Y .E E .QKVGK A Y.P- N TL.A L T
183 E.L V H.DQP V.I V R QT.YAT.D I DI.K.Y .E E .QKVGK A Y.P- N TL.A L T
282 E.L.G.V H Q V H . QT.YAT.E I DI GD E .QRVGK A H.P- K NS L T
284 E.L.G.V H Q V H . QT.YAT.E I DI GD E .QRVGK A H.P- K NS L T
331 D.L.N.VN H Q.A D.V S . S.QT.Y.T.E I DI ED DE .QRVGQ A H.P- K AS L T
452 E.L.N I H.SQ V QT.YAT.D I DI.E.Y .QD E .QRVSK.VSR L.P- K NS L T
512 E.L.N VEP K V Q YAT.E I DI.K.Y TVN.TD R .HKV.EQ Q H.N- TN L T.L
514 E.L.N VEP K V Q YAT.E I DI.K.Y TVN.TD R .HKV.EQ Q H.N- TN L T.L
639 E.P.N.I H Q V QT.YAT.D I DI KN E .QRVGK P H.P- K TS L T
1011 E.L.N.V H Q V QT.YAT.E I EK E .QRVGK P H.P-.R K AS L T
1014 E.L.N.V H Q V QT.YAT.E I EK E .QRVGK P H.P-.R K AS L T
V4 V5 *** *** ** * *** *** ** * NL43 GGEFFYCNST QLFNSTWFNS TWSTEGSNNT EGSD-TITLP CRIKQFINMW QEVGKAMYAP PISGQIRCSS NITGLLLTRD GG -NNNNG SEIFRPGGGD MRDNWR BAL .N V E VENN I R R PED.K T.V
171 .DTS R NWT GTNIKSTG.D N.VNGN .K.R.I.R R Q Q.V.N.L NS-S I
182 .T G G.YNST -YMPN.TASN SN S I .I R A.N.T.K .V .SLTS T
183 .T G G.YNST -YMPN.TASN SN S I .I R A.N.T.K .V .SLTS T
282 R TS K G.YMPT -YMPN.TESN S -SI I .I R.I E.N.T.K I V EGDTIK.D T
284 R TS K G.YMPT -YMPN.TESN S -SI I .I R.I E.N.T.K I V KGDTIK.D T
334 R TS G G T -YMPN.TKSN SS SI I .I R VE.N.T.N T IGTEQ.D T
452 R TS G T -YW.NATKSN SS S.F.IY IV Q A.N.T.K .V .SGQDGPK DLQTWRRRYE GQLE
512 .HTS G PWN -NTQ.S N-HTEN .IVH R Q P.V.N.V .N N STE E G F L
639 R TS G T - -RLFNSTEGN SS SF I .I R E.N.T.K A L .IDP.N T
1011 R TS G T -YMPN.TK.N SN SS I .I R A.N.T.K .V .EGS .D T
Trang 4A score of -6.96 was used as a cut off for R5 strains and a
score of -2.88 was used as a cutoff for X4 strains Most of
these scores were higher than -6.96 especially AP.17
sequence which showed a score of -3.58 clearly below the
cut-off score for X4 strains Thus, all the clones were
clearly predicted to be R5 tropic We also used the PSSM
(sinsi) matrix to analyze these sequences and these scores
were in the range of -7.5 to -11.53 This predicts high R5
tropism and a NSI phenotype for all the Indian isolates
examined Higher numbers of positively charged amino
acids (R/K/H) have been correlated with the likelihood of
CXCR4 use In all of the Indian sequences in this study,
lower numbers of positive charges, in the range of 5 to 7,
were found (Table 2) Thus, in an in-silico prediction
model, all of the V3–V5 sequences of HIV-1 Indian
iso-lates were predicted to show R5-tropism
Replication of subtype C chimeric viruses in
T-Lymphocyte cell lines
We first sought to determine whether the subtype C env
chimeras retained the lymphotropic properties of the
parental clone HIV-1NL4-3 by infecting the T cell line
A3.01 with the subtype C chimeric viruses and parental
virus, HIV-1NL4-3 The T-cell line A3.01 expresses CD4
and CXCR4 but no CCR5 Our results showed that the
parental X4 tropic HIV-1NL4-3 productively infected and
replicated in A3.01 over a 27-day infection period
However, the subtype C chimeras did not replicate in A3.01 cell line (Fig 2), suggesting that these chimeras were no longer T-cell line tropic, unlike the parental clone These results further demonstrated that replace-ment of the V3–V5 regions of envelope of HIV-1NL4-3 with subtype C envelope from infected patients was responsible for the lack of capacity to use CXCR4 as a coreceptor for these chimeric viruses These data also suggest that the integrated proviruses that were present
in these subtype C infected patient samples were not X4 tropic
Coreceptor utilization of chimeric subtype C viruses
We then determined the coreceptor utilization of the
sub-type C env chimeras (clone designation in Fig 1 and Table
1) using U373-MAGI indicator cell lines These cell lines express the CD4 receptor in conjunction with either CCR5
or CXCR4 as a coreceptor and can be infected with either macrophage tropic (R5) or lymphotropic (X4) viruses, respectively These cells contain an inducible β-galactosi-dase reporter driven by HIV-1 LTR that can be used as an indicator for entry The known SI isolate HIV-1NL4-3, NSI isolate HIV-1BaL, and primary isolates were used as
con-trols As shown in Table 3, all the subtype C env chimeras
were able to infect the U373-MAGI-CCR5 cell line but were unable to infect the U373-MAGI-CXCR4 cell line This data also correlates well with the insilico prediction
Table 2: Prediction of coreceptor usage by Position Specific Scoring Matrix (PSSM) bioinformatics tool that predicts coreceptor usage.
Patient/
Clone
AP.17/171,
173
AP.18/182,
183
AP.28/282,
284
AP.33/331,
334
AP.4/452,
454
AP.5/512,
514
AP.10/1011,
1014
Trang 5models and confirms the coreceptor usage of these
viruses This data suggests that the cloning of these
chime-ras yielded functional envelope regions that can bind
CD4/CCR5 and allow virus entry and production of early
viral genes As seen by the counts for infectivity of the
MAGI-CCR5 line, the chimeras showed considerable
dif-ferences in infectivity Some chimeras (171, 173)
demon-strated remarkably high levels of infectivity when
compared to other chimeras and primary isolate controls
indicating an increased rate of entry for these chimeras
The levels of entry using increasing virus counts (5000
and 10,000 RT counts) during infection showed increased
level of entry for the same chimeras This shows that the
envelope region of subtype C HIV-1 obtained from the
patient samples shows an R5 phenotype for the virus
infecting the patient These results also suggest that all the
chimeric viruses obtained could have had different rates
of entry when infecting target cells This can be attributed
to the differences in the V3 region of the subtype C
chimeric DNA, reflecting the differences found in patient samples This also adds to previous work done by others [4,32,33] showing that the coreceptor utilization of sub-type C HIV-1 could be predominantly R5 even late in infection
Syncytium inducing capacity of subtype C chimeras
We examined the syncytium-inducing ability of the
sub-type C env chimeras by infecting MT-2 cell lines with the
chimeric viruses Viruses that produce a greater than four syncytia per field were denoted as syncytium inducing (SI) phenotype and the viruses that did not induce any syncytia were called as non-syncytium inducing (NSI) phenotype
As shown in the Table 3, all of the subtype C V3–V5 region chimeras failed to produce any syncytia in MT-2 cells and therefore are of the NSI phenotype (similar to known R5 isolates HIV-1BaL) The control parental virus HIV-1NL4-3 that has a known SI phenotype produced significant levels
of syncytia (at least 10 per field of view) As expected, the
Replication of HIV-1 subtype C env V3–V5 region chimeras in T-lymphocyte (A3.01) cell line
Figure 2
Replication of HIV-1 subtype C env V3–V5 region chimeras in T-lymphocyte (A3.01) cell line A3.01 cells (1 × 106 cells/well)
were infected with equal amounts (RT counts) subtype C env chimeras (171 to 1014), parental HIV-1NL4-3 and HIV-1BaL Virus production was measured by reverse transcriptase (RT) assay in culture media harvested every 3 days and the cells fed with appropriate media The results are presented as cpm/ml ± SD of five separate triplicate experiments The subtype C chimeras were unable to replicate in A3.01 cell line
Viral Isolates
6 cp
Trang 6R5 viruses used as control also did not produce syncytia in
culture These data suggest that the envelope sequences
from subtype C infected patient samples render them less
cytopathic as compared to HIV-1NL4-3 We also confirmed
virus production in MT-2 cells and the syncytium
forma-tion correlated to virus producforma-tion in our partial env (V3–
V5) region chimeras (NSI) and R5 isolates as measured by
RT assay in the culture medium (not shown)
Replication of subtype C chimeras in primary peripheral
blood T-lymphocytes
Primary T-lymphocytes from peripheral blood express
CD4 and both chemokine receptors, CXCR4 and CCR5
Since PBL express both CCR5 and CXCR4, they are capable
of supporting the replication of both R5 and X4 tropic
viruses All the subtype C env chimeras, which did not
rep-licate in A3.01, were able to reprep-licate in PBL showing that
there was no inherent defect in their replication as a results
of the reciprocal insertion of the V3–V5 region from
patient samples (Fig 3) Although we have demonstrated
the entry of these chimeric viruses using the MAGI cell
experiments, the replication kinetics shown in Figure 3
confirm the capacity of these viruses to enter and replicate
well in culture
The replication kinetics of subtype C chimeras in PBL
showed that these chimeras replicated at a higher
efficiency as compared to subtype B chimeras (M5g, M7f, and M1c [34]) and subtype B isolates Close observation showed that chimeras 171 and 173 replicated and peaked much earlier in infection as compared to the other chime-ras The V3–V5 region of these chimeras came from a patient who demonstrated advanced disease (Table 1) [5] Although some chimeras (284 and 331) peaked relatively late in infection, they peaked at higher levels than the sub-type B chimeras All the subsub-type C chimeras also replicated
at levels higher than the subtype B primary isolates The replication data of the subtype C chimeras (Fig 3) corre-lated well with the rate of entry seen in the MAGI cell line experiments (Table 3) The chimeras that scored higher numbers in MAGI cell experiments (Table 3) peaked ear-lier in viral infection experiments (Fig 3) Comparative rates of entry of chimeras correspond with the peak of viral replication, where 171 and 173 with highest level of entry
in MAGI cells showed very early and high peaks and 284 and 331 chimeras with much lower level of entry showed lower and/or more delayed peaks It is interesting to note that chimeras 171, 173, 512 and 514, which retained the first proximal glycosylation site of the V3 region (Fig 1), peaked very early (Day 6–9) during replication (Fig 3) Comparison of the primary isolates of subtype B and sub-type C also showed that the subsub-type C primary isolates replicated better than the subtype B primary isolates These data suggest that the envelope V3 to V5 regions of
Table 3: Coreceptor usage by HIV-1 subtype C chimeras in U373-MAGI-CCR5 and U373-MAGI-CXCR4 cell lines.
MAGI-CCR5 MAGI-CXCR4
Infection counts → 5000 10000 5000 10000 Phenotype from
MT-2 CHIMERA_ No of blue cells No of blue cells
NSI – non-syncytium inducing, SI – syncytium inducing, - indicate no entry due to lack of blue cells.
Trang 7the subtype C influenced the rate of replication of HIV-1
in primary T-lymphocytes and determined the cellular
tropism Furthermore, there was a direct relationship
between higher viral replication (Fig 3) and advanced
dis-ease status of the patients (Table 1)
Replication of subtype C chimeras in primary
monocyte-derived macrophages
While primary monocyte derived macrophages (MDM)
express CD4 and CCR5 and a low level of CXCR4, they
support productive infection of R5 but not X4 viruses As
the subtype C chimeras showed a R5 phenotype (Table 3),
replication kinetics of these chimeras were evaluated in
MDM Figure 4 shows the replication kinetics of subtype C
chimeras in comparison with primary subtype B and C
controls The data clearly demonstrated that subtype C
chi-meras replicated better than subtype B viruses The rate of
replication of the subtype C chimeras in MDM also
corre-lated with the rate of entry of the chimeras in MAGI-CCR5
cell line, further supporting the hypothesis that the increase in the replication of these chimeras is due to increase in the rate of entry Both subtype B (2099) and subtype C (3041) primary R5 isolates replicated in MDM and subtype B dual tropic (X4/R5) virus (2101) also showed adequate replication in MDM In addition, com-parison of subtype B and subtype C primary isolates also showed that the subtype C primary isolates replicated better than subtype B primary isolates These data suggest that the V3 to V5 regions of subtype C influenced increased replication of HIV-1 in MDM
Discussion
We have provided evidence regarding the role of the
HIV-1 envelope V3–V5 regions from subtype C infected patients from India in virus entry, coreceptor utilization and replication efficiency in primary T-lymphocytes and macrophages in comparison with those of subtype B viruses Our data suggest that the reciprocal insertion of
Replication of HIV-1 subtype C env V3–V5 region chimeras in primary peripheral blood T-lymphocytes (PBL)
Figure 3
Replication of HIV-1 subtype C env V3–V5 region chimeras in primary peripheral blood T-lymphocytes (PBL) PBL (1 × 106
cells/well) were stimulated with PHA and infected with equal amounts (reverse transcriptase counts) of subtype C env V3–V5 region chimeras, subtype B env V3 region chimeras, primary subtype B isolates, primary subtype C isolates and parental
HIV-1NL4-3 Cells were fed every 3 days with appropriate medium and virus production was measured in the culture supernatant by
RT assay The data are presented as cpm/ml ± SD on triplicate experiments and are based on PBL from five different donors
Viral Isolates
6 cp
Trang 8HIV-1 subtype C infected patients' envelope V3 to V5
regions [5] into subtype B molecular clone, contributed to
utilization of CCR5 coreceptor (Table 3) as well as higher
levels of HIV-1 entry and replication efficiencies in
pri-mary T-lymphocytes (Fig 3) and MDM (Fig 4) The
higher viral replication efficiencies of R5 phenotype of the
chimeras correlated with advanced disease status of the
patients (Table 1) Taken together, the increased
replica-tion capabilities of HIV-1 subtype C in T-lymphocytes and
MDM may contribute to a high viral load, rapid disease
progression, and spread in infected individuals [35,36]
We have demonstrated that subtype C envelope V3–V5
region chimeras showed increased levels of virus entry
that correlated with an increased rate of replication in
pri-mary T-lymphocytes and MDM compared with subtype B
chimeras and subtype B primary isolates Careful
observa-tion indicates that chimeras with higher rate of entry
peaked earlier during infection in primary cells Unlike
the lymphotropic (X4) parental clone HIV-1NL4-3, the
subtype C env V3–V5 region chimeras were unable to
rep-licate in T lymphocyte cell lines A3.01 (Fig 2) and MT-2 (Table 3), suggesting that the chimeras had lost the T-cell line tropism of the parent clone NL4-3 because of recipro-cal insertion of the V3–V5 region from subtype C patient
samples In addition, all of the subtype C env chimeras
failed to produce any syncytia in MT-2 cells (Table 3), denoting NSI phenotypes, similar to the R5 but not
NL4-3 isolates Infection of UNL4-37NL4-3-Magi-X4 and UNL4-37NL4-3-Magi-R5 cell lines indicate that all our chimeric viruses and the control R5-tropic isolate HIV-1BaL, utilized the CCR5 core-ceptor, whereas the parental HIV-1NL4-3 utilized the CXCR4 coreceptor (Table 3) These results are consistent with earlier reports that showed reciprocal insertion of the V3 region of an R5 isolate into an X4 molecular clone altered the tropism of an X4 isolate to an R5 phenotype [10,19,23,28-30]
Our in-silico analysis of the V3 sequences of the subtype
C isolates from India predicted R5 tropism (Table 2) This
Replication of HIV-1 subtype C env V3–V5 region chimeras in primary blood monocyte-derived macrophages (MDM)
Figure 4
Replication of HIV-1 subtype C env V3–V5 region chimeras in primary blood monocyte-derived macrophages (MDM) MDM
(0.5 × 106 cells/well) were infected with equal amounts (RT counts) of subtype C env V3–V5 region chimeras, primary subtype
B isolates, primary subtype C isolates and HIV-1BaL Cells were fed every 3 days with appropriate medium and the virus pro-duction was measured in the culture supernatant by RT assay The data are presented as cpm/ml ± SD on triplicate experi-ments and are based on MDM from five different donors
0
2
4
6
8
10
12
14
171 173 182 183 282 284 331
221A 639 B-R5
C-R5 C-X4 BAL
Viral Isolates
5 cp
Trang 9also explains why these viruses replicated in primary
blood Tlymphocytes (Fig 3) but failed to replicate in the
T4 lymphocyte cell lines, A3.01 (Fig 2) and MT-2 because
primary T-lymphocytes express CCR5, whereas and A3.01
and MT-2 do not This data further supports the
predom-inance of R5 phenotype in subtype C infected patients
[37,38] and its maintenance during symptomatic AIDS
Several of the patients that exhibited advanced stages of
HIV disease (Table 1) also harbored R5 phenotype (Table
2, 3), rarely seen in subtype B infected adult patients In
addition, the chimeras from the patients with advanced
disease status (III, IV) replicated more efficiently than the
less advanced disease patients (I, II) [Table 1, Figs 3 and
4] Furthermore, it has also been found that the
percent-age of CD4 T cells expressing CCR5 in Indian adults is
higher than among Caucasian races [39] It is, therefore,
likely that due to the presence of this larger pool of CCR5
positive CD4 cells, the virus may not need a coreceptor
switch during disease progression
Our data showed that subtype C V3–V5 region
chime-ras replicated better in primary blood T-lymphocytes
(Fig 3) and MDM (Fig 4) compared with subtype B
chimeras, HIV-1NL4-3 and subtype B primary isolates In
general, there was a correlation between higher virus
entry, earlier replication peaks, and increased
replica-tion efficiencies in primary T-lymphocytes and MDM
Examination of sequence data published earlier on
these patients [5] and presented in Fig 1 and Table
show several features of subtype C chimeras, including
number of positive charges, amino acid sequence
vari-ation and number of glycosylvari-ation sites One striking
feature was that the chimeras from patient AP.17
(chi-meras # 171, 173) and AP.5 (chi(chi-meras # 512, 514)
retained the N-linked glycosylation site that includes
the first cysteine residue of V3 loop [10], whereas other
chimeras had mutations in this site, contributing to the
differences in replication efficiencies of these chimeras
(Fig 3) Our subtype V3–V5 region chimeras were in
close resemblance with subtype C (HIV-1C) with V3
sequences similar to HIV-1BAL (R5), as shown in Fig 1
The motif SIHIGPGRALYTTGEIIGDI that is important
for R5 tropism as seen for HIV-1BAL in Fig 1 was fairly
conserved in our subtype C chimeras contributing to R5
tropism Similarly subtype C chimeras V4–V5 region
sequences show similarity to subtype C sequence but
variability to subtype B (X4 and R5) sequences The
difference in amino acid sequences in V3 to V5 regions
in subtype C chimeras as compared to subtype B
sequences may be responsible for increased replication
efficiencies of subtype C chimeras Further studies on
site directed mutagenesis of the V3–V5 regions and
binding affinity of gp120 to CCR5 and/or gp120
incorporation into chimeric virions might pinpoint the
major difference in replication efficiencies
While a co-infection in vitro study with more fit subtype B
and less fit subtype C viruses indicates more fitness of
sub-type B over subsub-type C viruses [40], several in-vivo infection
studies on rhesus macaques have shown that HIV-1 subtype
C env chimeric viruses demonstrate greatly enhanced
infec-tivity [41-43] and replication efficiency as compared to sub-type B and E viruses [44,45] Our data on higher levels of HIV-1 entry and increased replication efficiencies of subtype
C chimeras compared with subtype B viruses is consistent
with the latter in vivo studies [41-43] Increased replication
efficiency of subtype C viruses has also been attributed to the presence of an extra NF-κB site in the LTR of subtype C viruses [46,47] However, these viruses have shown not only to replicate efficiently but to also be transmitted and spread more efficiently than other HIV-1 subtypes [1,9] During horizontal and vertical transmission subtype C viruses have been shown to spread more rapidly than other subtypes due to increased mucosal and vaginal shedding [36,48,49] This increased transmission cannot solely be explained due to increased LTR activity as increased shed-ding of virus and increased transmission may be attributed
to the envelope gene and others regions and functions of the virus
While various HIV-1 subtypes prevalent in different regions
of the world show variability in their replication kinetics and disease progression in infected individuals [32], HIV-1 subtype C is rapidly spreading and has already become the predominant subtype worldwide The data presented in this paper indicate that HIV-1 envelope V3–V5 region of subtype C contributes to increased rates of replication as compared to subtype B This would also explain higher viral loads and faster disease progression in patients infected with HIV-1 subtype C [35] as well as increased shedding of virus seen in subtype C infected population [36,48] Our data shows the HIV-1 subtype C envelope V3– V5 region may be one of the determinants of increased virus entry and replication These results provide another candidate gene to be responsible for the rapid spread of HIV-1 subtype C and its ability to dominate in populations that initially had higher incidence of other subtypes
Methods
Construction of HIV-1 Chimeras
Sixteen previously characterized envelope V3–V5 region sequences (Fig 1) from nine subtype C Indian isolates (Table 1) [5] were reciprocally substituted into pNL4-3 in
BglII-BglII sites flanking the region The 650 bp fragment
encompassing the V3–V5 region corresponds to amino acids 240–439 of gp120 of the HIV-INL4-3 sequence with the V3-loop positioned between amino acids 267 and 300 PCR primers were designed to amplify the V3–V5 region and to replace a similar region in pNL4-3 by engineering a
BglII site at the 3' end at position 7611 corresponding to all
isolates PCR amplification was carried out using Pfu
Trang 10polymerase The ~600 bp PCR product was digested with
BglII and cloned into pGEM (NL4-3) using the BglII sites
[50] The BglII-BglII fragment was reciprocally exchanged
into the pGEM plasmid containing the EcoRI-BamHI
fragment as there were no EcoRI or BamHI sites in this a
region in any Indian isolate except AP.5 which contained
an EcoRI site in the sequence The recombinant clones
were checked by digestion with BglII and the orientation
was confirmed by DNA sequencing using primer
(5'TCAACTGCTGTTAAATGGC3') Finally, the modified
EcoRI-BamHI fragment containing the sporadic subtype C
Indian isolate V3 to V5 region was reciprocally substituted
into pNL4-3 Two clones were obtained from each patient
sample and these are numbered with numerals of patient
identification number followed by clone number All the
clones were again checked by digestion with EcoRI/SalI
and BamHI and confirmed by sequencing.
Cell lines and primary cells
HeLa cells, U373-MAGI-CXCR4 and U373-MAGI-CCR5
cell lines were cultured in DMEM with 10% fetal bovine
serum (FBS) and penicillin-streptomycin (Invitrogen)
The MAGI cell media was also supplemented with 0.2 mg/
ml of G418, 0.1 mg/ml of hygromycin B and 1 μg/ml of
puromycin T-lymphocyte cell lines, A3.01 and MT-2 were
cultured in RPMI 1640 supplemented with 10% FBS and
penicillin-streptomycin Peripheral blood mononuclear
cells (PBMC) were obtained by single step density
gradi-ent cgradi-entrifugation with Ficoll-Hypaque (Amersham) from
whole blood of normal donors The blood collections
were made after informed consent, and were approved by
the Human Subject Ethical committees of International
Center for Genetic Engineering and Biotechnology
(ICGEB) and the Human Subjects Committee of the
Uni-versity of Arizona (Tucson, AZ) and were based on Indian
Council of Medical Research (ICMR) and National
Insti-tutes of Health (NIH) guidelines, respectively PBMC were
collected, washed twice with cold PBS and centrifuged at
1000 rpm for 10 min to avoid collecting platelets Primary
monocyte/macrophages and peripheral blood
lym-phocytes (PBL) were separated from PBMC using
mag-netic bead isolation protocols Monocytes were isolated
by CD14 magnetic bead isolation (Miltenyi biotech)
according to the manufacturer's protocols The cells
bound to the CD14 bead were used as primary
mono-cytes The primary monocytes eluted from the CD14
iso-lation columns were counted and plated in 48 well plates
at 1 × 106 cells/well in RPMI 1640 with 15% human AB
serum (Gemini biotech) and MCSF (Sigma) for 7 days
and were allowed to differentiate into macrophages in this
media The cells were fed every two days during
differen-tiation The cells collected as unbound flow through from
the CD14 bead isolation protocol were used as PBL PBL
were cultured in RPMI 1640 with 10% FBS and
penicillin-streptomycin PBL were stimulated with 5 μg/ml of PHA
for 24–48 h The stimulated cells were washed with PBS and resuspended in RPMI 1640 with 10% FBS and 20 U/
ml of recombinant human IL-2 (Invitrogen)
DNA Transfections
Subtype C chimeric proviral DNAs were transfected in HeLa cells by electroporation as described before [34] or
by Lipofectamine 2000 (Invitrogen) [51] For the Lipo-fectamine method, HeLa cells were grown in DMEM with 10% FBS and penicillin-streptomycin to about 80% con-fluency The cells were then split and counted and plated
in a 6-well plate at 105 cells/well in DMEM with 10% FBS without antibiotics The cells were transfected the next day with 3 μg DNA in DNA-lipofectamine complexes as per manufacturer's procedure Chimeric viruses were har-vested by collecting culture supernatant from the wells 72 hrs post-transfections Virus production was measured by
a reverse transcriptase (RT) assay [34,51]
Infections
A3.01 cells (2 × 106), MT-2 (2 × 106), PBL (1 × 106) and MDM (0.5 × 106) per well were cultured and infected with
equal amounts of subtype C env region chimeras, subtype
B V3 region chimeras (M5g, M7f, M1c) [34,50], subtype B and C primary isolates, and virus production was meas-ured every 3 days We used two subtype C (3041-R5 and 5441-X4) and two subtype B (2099-R5 and 2101-X4/R5) primary isolates that were obtained from AIDS Research and Reference Reagent Program as controls Briefly, viruses were adsorbed in A3.01 cells, MT-2 and PBL for 90 min in serum free media at 37°C and 5% CO2 After incu-bation, 500 μl of appropriate media containing serum and antibiotics were added MDM were infected in media containing serum and polybrene (8 μg/ml) incubated at 37°C and 5% CO2 for 16 hrs After incubation, the mac-rophages were washed in PBS to remove polybrene and resuspended in macrophage culture media
Coreceptor utilization
U373-MAGI-CXCR4 and U373-MAGI-CCR5 cell lines were plated at ~6 × 104 cells/well in 24 well plate in com-plete DMEM with G418-hygromycin-puromycin Both cells lines were infected with 5,000 and 10,000 cpm (RT assay counts) of chimeric subtype C virus and primary iso-late controls diluted in a total volume of 300 μl of com-plete DMEM (without antibiotics) with DEAE-dextran (final concentration 20 μg/ml) Two hours post-adsorbtion, 1.5 ml of fresh MAGI media was added To assess the rate of entry, 40 h post-infection, the medium was removed and cells were fixed in 1% formaldehyde and 0.2% glutaraldehyde for 5 min Then, the cells were washed with PBS and stained for 2 hrs in staining solution containing 0.2 M potassium ferricyanide, 0.2 M potassium ferrocyanide, 2 M MgCl2 and 40 μg/ml X-Gal After staining, the cells were washed in PBS and resuspended in